Production Technology for Recombinant IVIg Organization: GigaGen Inc. PI: David S. Johnson, Ph.D. The Specific Aim of this SBIR Phase I project is to develop a natural repertoire antibody protein expression system that will form the basis of a recombinant intravenous immunoglobulin (rIVIg) therapeutic product. IVIg is a pool of proteins isolated from the sera of thousands of donors. The FDA has approved IVIg therapy for six indications, including idiopathic (immune) thrombocytopenic purpura (ITP), Kawasaki's vasculitis, B cell chronic lymphocytic leukemia (CLL), and primary immunodeficiencies (Orange et al., 2006). IVIg sales are $7 billion worldwide and growing at 8-10% per year, due to an aging population and ever-expanding off-label modalities (Taylor & Shapiro, 2013). Unfortunately, current methods for IVIg production threaten continued expansion of IVIg therapy because of supply chain risk, impurities and contamination, and batch-to-batch variation. In this Phase I project, we will take steps to demonstrate that we can express GigaMuneTM natural human repertoire DNA libraries a stable yeast expression system. Analogous to Genentech 30 years ago (Russo 2003), our primary technology innovation is to use natural Ig repertoire DNA libraries expressed in a humanized Pichia yeast production system (Li et al., 2006) to replace a resource-limited biological drug with a recombinant alternative. To make rIVIg, we will first use GigaGen GigaMuneTM technology to capture and re- create expressed Ig repertoires from >1000 blood donors. GigaMuneTM uses advanced microfluidics and genomics to generate RT-PCR libraries from millions of single cells per donor, with native IgG subtypes and pairing between heavy and light chain. We will then stably express the DNA repertoires en masse in engineered Pichia to produce massively polyclonal engineered rIVIg protein product. Our rIVIg will have natural repertoire genetics, engineerable content, programmable glycosylation, low production cost, and consistent and predictable production. We will accomplish the Specific Aim by performing the following tasks: (i) Engineer a system for subcloning GigaLink(tm) DNA libraries en masse with native IgG isotype intact; (ii) Optimize GigaLink(tm) DNA library delivery and stable display in a Pichia yeast production system; and (iii) Use next-generation sequencing (NGS) and Ig assays to assess several cell passages for uniformity and isotype content. We will be successful if we achieve the following metrics: (i) Use NGS to show that the Ig clone frequencies of the >107 diversity GigaLink(tm) libraries are maintained when subcloned and stably expressed in the yeast production system (linear regression; a=0.05, power=0.8); and (ii) Use NGS and antigen binding assays to demonstrate <10% CV between cell passages and time points (one-proportion z-test; a=0.05, power=0.8). Phase I will demonstrate that we can reproducibly produce high-diversity GigaLink(tm) protein libraries in a yeast expression platform. In Phase II, we will take steps to build a GMP production facility and perform toxicology and pharmacokinetic studies on our rIVIg preparations. At first, rIVIg will simply substitute for conventional IVIg, especially for patients who are deficient in antibodies (i.e., hypogammaglobulinemia or humoral deficiencies). Later, our ability to engineer the content of the DNA library will open up broad new applications, such as IgA deficiency and polyclonal anti-tumor therapeutics.